Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
  • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

Definitions

• This invention relates to proteases derived from strains of Bacillus sp. More specifically, the invention is directed towards a novel alkaline protease derived from a strain of Bacillus sp. PD498, as well as isolated biologically pure cultures of Bacillus sp. PD498.
• the invention is directed towards a process for the preparation of the protease, detergent additives and detergent compositions comprising the protease of the invention, and the use of this protease in washing processes.
• Enzymes used in washing formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures hereof. Commercially most important are proteases.
• Detergent proteases have been developed by isolation of proteases found in nature followed by testing in detergent formulations. Most detergent proteases are obtained from members of the genus Bacillus. Currently new types of proteases enter the market, offering the possibility of giving a better cost/performance ratio at various specified conditions.
• Examples of commercial protease products are ALCALASETM, ESPERASETM and SAVINASETM, all supplied by Novo Nordisk A/S, Denmark. These and similar enzyme products from other commercial sources are active in detergent solutions, i.e. at pH values in the range of from 8 to 11 and in the presence of sequestering agents, surfactants and bleaching agents such as sodium borate.
• the ALCALASETM protease is produced by strains of the species Bacillus licheniformis.
• the ESPERASETM and SAVINASETM proteases are obtained by cultivation of strains of alkalophilic Bacilli.
• novel detergent proteases are provided.
• the invention provides a protease having an apparent molecular weight of 34 kD as determined by SDS-PAGE, a pI of approximately 9.3, a pH optimum in the range of pH 9-11 determined at 25°C (with casein as substrate), a temperature optimum in the range of 40-55°C determined at pH 9.5 (with casein as substrate), and immunochemical properties identical or partially identical to those of a protease derived from Bacillus sp. PD498, NCIMB No. 40484.
• the invention provides a protease having an apparent molecular weight of 34 kD as determined by SDS-PAGE, a pl of approximately 9.3, a pH optimum in the range of pH 9-11 determined at 25°C (with casein as substrate), a temperature optimum in the range of 40-55°C determined at pH 9.5 (with casein as substrate), and being obtainable from a strain of Bacillus sp. PD498, or from another host organism carrying the gene encoding a protease having immunochemical properties identical or partially identical to those of the protease derived from Bacillus sp. PD498.
• the invention provides an isolated biologically pure culture of a strain of Bacillus sp. PD498.
• a strain of Bacillus sp. PD498 in a more specific aspect, a strain of Bacillus sp. PD498.
• Bacillus sp. PD498, NCIMB No. 40484, or a mutant or a variant thereof, is provided.
• the invention provides a process for the preparation of the protease, which process comprises cultivation of a protease producing strain of Bacillus sp. PD498 in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
• Bacillus sp. PD498, NCIMB No. 40484, or a mutant or a variant thereof, or another host organism carrying the gene encoding a protease having immunochemical properties identical or partially identical to those of the protease derived from Bacillus sp. PD498, is cultivated.
• the use of the enzyme as detergent enzyme is claimed.
• the invention provides detergent additives and detergent compositions comprising the protease of the invention.
• the invention relates to the use of the enzyme of the invention in washing processes. BRIEF DESCRIPTION OF DRAWINGS
• Fig. 1 shows the relation between temperature and the proteolytic activity of an enzyme according to the invention (the enzyme preparation obtained according to Ex. 1 , with 2% casein as substrate, determined at pH 9.5; Buffer, D Buffer + 0.1% STPP);
• Fig. 2 shows the relation between pH and the proteolytic activity of an enzyme according to the invention (the enzyme preparation obtained according to Ex. 1 , with 1% casein as substrate, determined at 25°C); and
• Fig. 3 shows the washing performance of an enzyme of the invention compared to a commercial detergent enzyme ( ⁇ the enzyme preparation obtained according to Ex. 1 ; O SavinaseTM, Novo Nordisk A/S, Denmark).
• the novel microorganism of the invention able to produce an enzyme of the invention, was isolated from a soil sample.
• Bacillus sp. PD498 has been deposited according to the Budapest Treaty at NCIMB, under No. 40484.
• the microorganism of this invention is an aerobic, spore forming bacterium belonging to the genus Bacillus. Morphologically it can be described as motile rods with a diameter of 0.7 - 0.9 micron, and a length of 2 - 3 micron.
• the spores are round to ellipsoid, slightly swelling the sporangium, subterminal to terminal.
• Optimum temperature for growth is within 25-37°C
• optimal pH for growth is within 7-9, no growth at pH 9.7, and no growth at 50°C.
• the micro organism forms yellow colonies, round and smooth, on nutrient agar slants, and no diffusion of pigment into the agar is observed.
• the cellular characteristics of the strain Bacillus sp. PD498 of the invention have been compared to those of the known Bacillus sp. type strains deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 B, D-3300 Braunschweig, Germany.
• the microorganism of the invention can be cultivated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrients, the medium being composed in accordance with the principles of the known art.
• Suitable carbon sources are carbohydrates such as sucrose, glucose and starch, or carbohydrate containing materials such as cereal grain, malt, rice and sorghum.
• the carbohydrate concentration incorporated in the medium may vary widely, e.g. up to 25% and down to 1-5%, but usually 8-10% will be suitable, the percentages being calculated as equivalents of glucose.
• the nitrogen source in the nutrient medium may be of inorganic and/or organic nature. Suitable inorganic nitrogen sources are nitrates and ammonium salts. Among the organic nitrogen sources quite a number are used regularly in fermentation processes involving the cultivation of bacteria. Illustrative examples are soybean meal, cotton seed meal, peanut meal, casein, corn, corn steep liquor, yeast extract, urea, and albumin. In addition, the nutrient medium should also contain usual trace substances.
• the novel Bacillus species of this invention is slightly alkalophilic. Therefore, the cultivation is preferably conducted at slightly alkaline pH values, which can be obtained by addition of suitable buffers such as sodium bicarbonate, pH 9.0, after sterilization of the growth medium.
• suitable buffers such as sodium bicarbonate, pH 9.0
• the rate of aeration is similar to that used in conventional tank fermentation.
• liquid enzyme concentrates may be produced by removal of coarse material from the broth and, if desired, concentration of the broth by evaporation at low temperature or by ultrafiltration. Finally, preservatives may be added to the concentrate.
• Solid enzyme preparations may be prepared from the purified and/or concentrated broth by precipitation with salts, such as Na 2 SO 4 or water-miscible solvents, such as ethanol or acetone. Removal of the water in the broth by suitable drying methods, such as spray-drying, may also be employed.
• salts such as Na 2 SO 4
• water-miscible solvents such as ethanol or acetone.
• proteolytic activity is determined with casein as substrate.
• Casein Protease Unit is defined as the amount of enzyme liberating 1 mM of primary amino groups (determined by comparison with a serine standard) per minute under standard conditions, i.e. incubation for 30 minutes at 25°C and pH 9.5.
• a folder AF 228, describing the analytical method, is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
• the enzyme of the invention is a novel detergent protease. It is an alkaline protease, obtainable by cultivation of a microorganism of the invention, preferably Bacillus sp. PD498, NCIMB No. 40484, or a mutant or a variant thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts.
• the enzyme can also be obtained by recombinant DNA-technology.
• the protease of the invention can be described by the following characteristics.
• the protease of the invention has an apparent molecular weight of 34 kD when determined by SDS-PAGE. A pl of approximately 9.3 was determined by isoelectric focusing on LKB Ampholine ® PAG plates. The protease activity is inhibited by PMSF, a serine proteinase inhibitor. EDTA and soybean-protein inhibitor do not influence the protease activity.
• the temperature activity relationship is presented on Fig. 1.
• the activity was determined with 2% casein as substrate at pH 9.5 in the presence (white squares) and absence (black squares) of 0.1 % sodium tripolyphosphate (STPP, a common ingredient in many commercial detergents).
• STPP sodium tripolyphosphate
• the assay for proteolytic activity described previously was used with the modification that the incubation temperature was varied in the interval of from 15 to 70°C. It appears from the figure that the enzyme possesses proteolytic activity from 15°C to 70°C, and has a temperature optimum in the range of from 40-55°C, around 50°C.
• the protease of the invention has immunochemical properties identical or partially identical (i.e. at least partially identical) to those of a protease derived from the strain Bacillus sp. PD498, NCIMB No. 40484.
• the immunochemical properties can be determined immunologically by cross-reaction identity tests.
• the identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectrophoresis according to N. H. Axelsen; Handbook of Immunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications (1983), chapters 5 and 14.
• the terms "antigenic identity” and “partial antigenic identity” are described in the same book, Chapters 5, 19 and 20.
• Monospecific antiserum was generated according to the above mentioned method by immunizing rabbits with the purified protease of the invention.
• the immunogen was mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week.
• Antiserum was obtained after a total immunization period of 8 weeks, and immunoglobulin was prepared therefrom as described by N. H. Axelsen, supra.
• the detergent composition of the invention may comprise one or more surfactants, which may be of an anionic, non-ionic, cat-ionic, amphoteric or zwitterionic type, or a mixture of these.
• anionic surfactants are linear alkyl benzene sulfonates (LAS), alkyl sulfates (AS), alpha olefin sulfonates (AOS), alcohol ethoxy sulfates (AES) and alkali metal salts of natural fatty acids.
• non-ionic surfactants are alkyl polyethylene glycol ethers, nonylphenol polyethylene glycol ethers, fatty acids esters of sucrose and glucose, and esters of polyethoxylated alkyl glucoside.
• the detergent composition of the invention may also contain other detergent ingredients known in the art such as builders, bleaching agents, bleach activators, anti-corrosion agents, sequestering agents, anti soil-redeposition agents, perfumes, stabilizers for the enzymes and bleaching agents, formulations aids, optical brighteners, foam boosters, chelating agents, fillers, fabric softeners, etc.
• the detergent composition of the invention may be formulated substantially as described in Falbe, J. [Falbe, J.; Surfactants in Consumer Products. Theory, Technology and Application; Springer Verlag 1987, vide in particular the section entitled "Frame formulations for liquid/powder heavy-duty detergents"].
• the detergent composition of the invention may contain the enzyme preparation in an amount corresponding to 0.0005-0.5 CPU of the proteolytic enzyme per litre of washing liquor.
• the detergent compositions of the invention can be formulated in any convenient form, such as powders, liquids, etc.
• the detergent composition of the invention may advantageously include one or more other enzymes, e.g. Iipases, amylases, cellulases, oxidases, and/or peroxidases, conventionally included in detergent compositions.
• other enzymes e.g. Iipases, amylases, cellulases, oxidases, and/or peroxidases, conventionally included in detergent compositions.
• the protease of the invention may be included in a detergent composition by adding separate additives containing the detergent protease, or by adding a combined additive comprising different detergent enzymes.
• the additive of the invention can be formulated e.g. as granulates, liquids, slurries, etc.
• Preferred detergent additive formulations are non-dusting granulates, liquids, in particular stabilized liquids, slurries, or protected enzymes. Dust free granulates may be produced e.g. according to GB Patent Publication No. 1 ,362,365 or US Patent No. 4,106,991 , and may optionally be coated by methods known in the art.
• the detergent enzymes may be mixed before or after granulation.
• Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as e.g. propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid, according to established methods. Other enzyme stabilizers are well known in the art.
• Protected enzymes may be prepared according to the method disclosed in EP Patent Publication No. 238,216.
• Bacillus sp. PD498 was cultivated at 25°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml of medium of the following composition (per litre): Potato starch 100 g
• the starch in the medium is liquified with ⁇ -amylase, and the medium is sterilized by heating at 120°C for 45 minutes.
• the pH of the medium is adjusted to 9.0 by addition of 10 ml of a 1 M solution of sodium bicarbonate.
• protease was purified by a conventional chromatographic method.
• the tests were performed at enzyme concentrations between 0.02 and 0.5 mg of enzyme protein per litre.
• ⁇ R wash performance differential remission

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• 1992
  • 1992-05-27 DK DK92702A patent/DK70292D0/da not_active Application Discontinuation
• 1993
  • 1993-05-26 US US08/325,386 patent/US5597720A/en not_active Expired - Lifetime
  • 1993-05-26 KR KR1019940704053A patent/KR100284246B1/ko not_active IP Right Cessation
  • 1993-05-26 WO PCT/DK1993/000183 patent/WO1993024623A1/en active IP Right Grant
  • 1993-05-26 DE DE69328487T patent/DE69328487T2/de not_active Expired - Lifetime
  • 1993-05-26 EP EP93912668A patent/EP0652947B1/en not_active Expired - Lifetime
  • 1993-05-26 DK DK93912668T patent/DK0652947T3/da active
  • 1993-05-26 AT AT93912668T patent/ATE192190T1/de not_active IP Right Cessation
  • 1993-05-26 JP JP50009894A patent/JP3309985B2/ja not_active Expired - Lifetime
  • 1993-05-26 ES ES93912668T patent/ES2148230T3/es not_active Expired - Lifetime
• 1994
  • 1994-11-25 FI FI945543A patent/FI117759B/fi not_active IP Right Cessation

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